Application of PINK1 / Parkin in cognitive impairment by adjusting mitochondria

Abstract

The invention discloses application of PINK1 / Parkin in cognitive impairment by adjusting mitochondria, and relates to the technical field of biological medicine, and the technical key points are as follows: the PINK1 / Parkin can mediate the autophagy mechanism of the mitochondria, inhibit excessive mitochondrial autophagy generated by the cognitive impairment caused by aluminum exposure, maintain the steady state balance of the mitochondria in the body of a patient, and inhibit the mitochondrial autophagy caused by aluminum exposure. The excessive formation of ROS is inhibited, and the progress of cognitive impairment of a patient is slowed down.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of PINK1 / Parkin in cognitive dysfunction by regulating mitochondria. Background Art

[0002] Aluminum is a neurotoxic element that has been shown to play an important role in the degeneration of nerve cells in the brains of humans and experimental animals. Experiments have shown that under long-term exposure to aluminum, aluminum accumulates in all areas of the rat brain, with the most significant accumulation in the hippocampus. Therefore, it is speculated that the hippocampus is an important target area for aluminum neurotoxicity. Epidemiological studies have found that there is a positive correlation between aluminum concentrations in the environment (especially aluminum concentrations in drinking water) and cognitive dysfunction. In vitro studies have found that most workers engaged in aluminum casting operations have some memory loss. However, the early molecular and cellular events of its neurotoxicity are still unclear.

[0003] To this end, the present invention aims to provide an application of PINK1 / Parkin in cognitive dysfunction by regulating mitochondria to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide the application of PINK1 / Parkin in cognitive dysfunction by regulating mitochondria. The PINK1 / Parkin can mediate the mitochondrial autophagy mechanism, inhibit excessive mitochondrial autophagy caused by cognitive dysfunction caused by aluminum exposure, maintain mitochondrial homeostasis in patients, and inhibit excessive formation of ROS, thereby alleviating patients' cognitive dysfunction and improving their quality of life, providing new ideas for the treatment of cognitive dysfunction.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention provides the use of PINK1 / Parkin in cognitive dysfunction by regulating mitochondria. PINK1 / Parkin can be used to prepare a drug for treating cognitive dysfunction caused by excessive aluminum intake. PINK1 / Parkin can regulate the mitochondrial autophagy mechanism, inhibiting the excessive mitochondrial autophagy caused by aluminum exposure-induced cognitive dysfunction, maintaining mitochondrial homeostasis in patients, and inhibiting the excessive formation of ROS, thereby alleviating cognitive dysfunction in patients.

[0007] Maltoaluminum exposure can increase the production of ROS in the nervous system. Excessive ROS attack mitochondria, stimulate oxidative stress, lead to mitochondrial structural damage and dysfunction, and reduce the mitochondrial energy supply capacity. At the same time, damaged mitochondria trigger the PINK1 / Parkin-mediated autophagy mechanism, resulting in excessive mitophagy and further reducing the number of mitochondria. Mitochondria play a core regulatory role in apoptosis. Therefore, maltoaluminum exposure can affect the apoptosis of nerve cells by damaging and altering mitochondrial structure and function and reducing the number of mitochondria in the patient's brain and neural tissue.

[0008] Compared with the existing technology, this solution has the following beneficial effects:

[0009] The PINK1 / Parkin of the present invention can mediate the mitochondrial autophagy mechanism, inhibit excessive mitochondrial autophagy caused by cognitive dysfunction due to aluminum exposure, maintain mitochondrial homeostasis in patients, and inhibit excessive formation of ROS, thereby alleviating patients' cognitive dysfunction, improving their quality of life, and enabling them to better complete daily activities, providing new ideas for the treatment of cognitive dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the action route of the mitochondrial autophagy pathway in rats after maltodextrin aluminum poisoning in an embodiment of the present invention;

[0011] Figure 2 Schematic diagram of the weight results of rats in each group before and after exposure to aluminum maltolate in the present invention, including the control group (CG), low-dose group (LG), medium-dose group (MG), and high-dose group (HG); mean ± SD. n = 8; *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG;

[0012] Figure 3 Schematic diagram of the Morris water maze test results of rats in each group exposed to aluminum maltol in an embodiment of the present invention, wherein the escape latency of the four groups of rats in A gradually shortened; in B, the number of rats crossing the platform gradually decreased with the increase in the dose of aluminum maltol exposure; in C, the number of rats crossing the platform quadrant gradually decreased with the increase in the dose of aluminum maltol exposure; mean ± SD. n = 8; aP < 0.05 vs. the same group on the first day; bP < 0.05 vs. the same group on the second day; cP < 0.5 vs. the same group on the third day; dP < 0.05 vs. the same group on the fourth day; *P < 0.05, **P < 0.01 vs. CG; #P < 0.05, ##P < 0.01 vs. LG;

[0013] Figure 4 Schematic diagram of the water maze trajectory diagram of each group of rats after exposure to maltodextrin aluminum in the embodiment of the present invention;

[0014] Figure 5 Schematic diagram of the ROS and GSH-Px results after maltol aluminum exposure in each group of rats in the embodiment of the present invention, wherein A: ROS immunofluorescence staining, DAPI, diaminobutylphenylindole, comparison of ROS expression characteristics in the cerebral cortex of the four groups of rats; after DAPI staining, the nuclei of neurons in the cerebral cortex of the four groups were uniformly blue; after ROS staining, the cells appeared uniformly red, and the red fluorescence intensity gradually increased with the gradual increase of aluminum exposure dose; B: The relative expression of ROS gradually increased with the gradual increase of aluminum exposure dose; C: The relative expression of GSH-Px gradually decreased with the gradual increase of aluminum exposure dose; (scale bar = 50 μm); mean ± SD. n = 8; *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG;

[0015] Figure 6 Schematic diagram of the mitochondrial electron microscopy results of rats in each group after exposure to aluminum maltophenol in the embodiment of the present invention, wherein A is the result of mitochondrial electron microscopy examination of the control group; B is the result of mitochondrial electron microscopy examination of the low-dose group; C is the result of mitochondrial electron microscopy examination of the medium-dose group; D is the result of mitochondrial electron microscopy examination of the high-dose group;

[0016] Figure 7 is the Na of each group of rats after exposure to maltol aluminum in the embodiment of the present invention + -K + --ATPase and Ca 2+ -Mg 2+ -ATPase results diagram, where Na in A + -K + -ATP activity gradually decreases; Ca in B 2+ -Mg 2+ -ATP activity gradually decreased; mean ± SD, n = 8, *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG;

[0017] Figure 8Schematic diagram of the results of mitophagy proteins in each group of tats after exposure to maltoaluminum in the embodiment of the present invention, wherein the band of protein PPINK1 in A becomes thicker and the relative expression gradually increases; the band of protein Parkin in B becomes thicker and the relative expression gradually increases; the band of protein P62 in C becomes thinner and the relative expression gradually decreases; the band of protein LC3BII. / LC3BI. in D becomes thicker and the ratio of LC3BII. / LC3BI. gradually increases; the band of protein COXIV. in E becomes thinner and the relative expression gradually decreases; mean ± SD. n = 8, *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG; & P < 0.05 vs MG;

[0018] Figure 9 Schematic diagram of the results of the corresponding genes of mitochondrial autophagy proteins in each group of rats after exposure to maltodextrin aluminum in the embodiment of the present invention, wherein the relative expression of the gene mPINK1 in A gradually increased; the relative expression of the gene mParkin in B gradually increased; the relative expression of the gene mP62 in C gradually decreased; the relative expression of the gene mLC3B in D gradually increased; the relative expression of the gene mCOXIV. in E gradually increased; mean ± SD. n = 8, *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG; &P < 0.05, &&P < 0.01 vs MG;

[0019] Figure 10 Schematic diagram of the corresponding genes of the apoptosis results after each group of rats were exposed to maltoaluminum in the examples of the present invention, wherein the relative expression of the gene mBcl-2 in A gradually decreased; the relative expression of the gene mBax in B gradually increased; the relative expression of the gene mCyt-c in C gradually increased; the relative expression of the gene mCaspase3 in D gradually increased; mean ± SD. n = 8, *P < 0.05, **P < 0.01 vs CG; #P < 0.05, ##P < 0.01 vs LG; &P < 0.05, &&P < 0.01 vs MG. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0021] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0022] Example:

[0023] 1. Materials and Methods

[0024] Reagents

[0025] AlCl3·6H2O (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.); Maltolate (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-PINK1 antibody (ab186303), mouse anti-Parkin1 antibody (ab77924), rabbit anti-SQSTM1 / p62 antibody (ab109012), rabbit anti-COXIV antibody (ab202554), and rabbit anti-GAPDH antibody (ab181602) (Abcom); rabbit anti-LC3B antibody (ET1701-65), horseradish peroxidase-conjugated goat anti-rabbit II antibody (9HA1001) (Hua'an Biotechnology Co., Ltd.); horseradish peroxidase-conjugated goat anti-mouse II antibody (S0002, Affinity); SDS-PAGE gel preparation kit (PG112) (Shanghai Yasi Biopharmaceutical Technology Co., Ltd.); UV spectrophotometer (Shanghai Xianke Instrument Co., Ltd.); and water maze experimental equipment (Jiangsu Saiang Biotechnology Co., Ltd.).

[0026] 1.2. Experimental Animal Grouping

[0027] Thirty-two healthy male Sprague-Dawley rats (6 weeks old, specific pathogen-free (SPF) grade) were divided into several groups for treatment and fed a regular diet with unrestricted access to food and water. The rats were randomly divided into four groups based on body weight: a control group (CG) (0 μmol / kg), a low-dose group (LG) (10 μmol / kg), a medium-dose group (MG) (20 μmol / kg), and a high-dose group (HG) (40 μmol / kg). Each group consisted of eight animals. Specific protocol: A rat exposure model was established by intraperitoneal injection of maltodextrin. The four groups of rats were each administered 1 mL / kg body weight. The control group was administered physiological saline solution (equivalent to a maltodextrin exposure dose of 0 μmol / kg), while the low-, medium-, and high-dose groups were administered maltodextrin solutions at concentrations of 10, 20, and 40 μmol / kg body weight, respectively, five days per week. The drug was discontinued for two days. Exposure lasted for three consecutive months. During the exposure period, the rats' feed and water intake were observed daily and replenished promptly. Their mental state and growth were observed weekly, and their body weights were measured and recorded.

[0028] 1.3. Design and preparation of exposure doses of aluminum maltolate

[0029] In 2011, the World Health Organization and the Food and Agriculture Organization of the United Nations (WHO / FAO) provisionally revised the acceptable weekly intake for aluminum to 2,000 mg / kg body weight / week. This means that adults should consume no more than 0.283 mg / kg body weight of aluminum per day. Based on the body surface area ratio of human to animal dose equivalents, rats ingest 6.3 times the equivalent amount of aluminum as humans. Therefore, the oral dose of Al for rats is 1.785 mg / kg body weight. Based on the dosage for different routes of administration, the intraperitoneal dose of Al for rats is 0.535 mg / kg body weight. The molar mass of Al is 26.98 g / mol, so the Al ion concentration is approximately 19.85 μmol / kg. Based on experience from previous experiments, toxic doses were set at low, medium, and high doses: 10 μmol / kg, 20 μmol / kg, and 40 μmol / kg. A control group was also established, with a toxic dose of 0 μmol / kg. The solution preparation method followed the previous experiment. First, 80, 40, and 20 mmol / L aluminum chloride solutions were prepared in distilled water, and 240, 120, and 60 mmol / L maltol solutions were prepared in PBS. The 80 mmol / L aluminum chloride solution was then mixed with the 240 mmol / L maltol aqueous solution in equal volumes to obtain a 40 mmol / L aluminum maltol solution. Equal volumes of the 40 and 20 mmol / L aluminum chloride aqueous solutions were mixed with the 120 and 60 mmol / L maltol solutions to obtain 20 mmol / L and 10 mmol / L aluminum maltol solutions. The pH was adjusted to approximately 7.4 with NaOH solution to prepare the aluminum maltol solution and use it without precipitation. This study has received research ethics approval from Jining Medical College (name of the approving institution), and the approval certificate / certificate is available upon request.

[0030] Morris water maze training and testing

[0031] Referring to other experiments, this experiment consists of two parts: (1) Spatial navigation test: The Morris water maze was divided into four equal parts, and the escape platform was located in the designated quadrant. On the day before the experiment officially started, the platform was visible and the rats were introduced to the water maze for 2 minutes to adapt to the environment of the maze. To officially start the experiment, the platform was placed about 2 cm underwater, and the rats were placed into the water from the four quadrants every day, facing the pool wall. The order of the launch quadrants was different on each of the five days. The time required for the rats to locate the platform from the time they entered the water, as well as the path they swam, were recorded. The average time required to find the platform in the four quadrants was determined as the escape latency. If the rats failed to find the platform within 120 seconds, they were guided to the platform and allowed to stay on the platform for 10 seconds. In this case, the escape latency was recorded as 120 seconds. (2) Spatial exploration experiment: On the sixth day, the underwater platform was removed from the maze and the rats were placed into the water from the designated quadrant. The frequency with which the rats crossed the area where the platform was located and the quadrant in front of the platform was recorded within 120 seconds.

[0032] 1.5. Novel Object Recognition Experiment

[0033] During the experiment, the test recognition time was set to 5 minutes. If the test time is set too long, the rat's ability to recognize objects will be weakened because as the test time increases, the rat will feel more familiar with the familiar object. One of the objects was replaced with another obviously different object (C), and the rat was placed in the open box according to the above method. The test time was set to 5 minutes, and the rat's discrimination time around the two objects was recorded. It was recorded as the new object time (new, N) and the familiar object time (familiar, F). The ability of the rat to explore new items was evaluated by calculating the discrimination index of exploring new objects. Discrimination index (DI) = (NF) / (N+F) × 100%. Preference index (PI) is used to measure memory preference, PI = [N / (N+F)] × 100%. Discrimination index and preference index are used to evaluate the cognitive and memory abilities of rats.

[0034] 1.6. Sample preparation by transmission electron microscopy (TEM)

[0035] After deep sedation, the rats were euthanized and their brain tissues were quickly extracted according to standard experimental procedures, while being careful to prevent any damage to the neural tissue. The cortex was separated from the hippocampus, and both ends of the hippocampus were removed, leaving only a small piece of tissue about 0.5 cm wide in the middle of the hippocampus. It was rinsed with double distilled water for 2-3 seconds to remove the blood remaining on the surface. It was fixed with a toothpick in a pre-cooled fixative solution (2% glutaraldehyde) and stored in a refrigerator at 4%. The tissue was rinsed with buffer for 2-3 hours, the solution was changed 4 times, and the tissue was fixed with osmium acid (1% OSO4Fix the tissue for no more than 2 hours, dehydrate using an acetone series, each step lasting 5-15 minutes, immerse in a mixture of embedding medium and acetone, allow to rest at ambient temperature for 2-4 hours, embed in epoxy resin, immerse at 40°C for 24 hours, and polymerize at 60°C for 48 hours. Use an LKB ultramicrotome; stain with uranyl acetate and lead citrate, repeating for 15-30 minutes each time; and observe using a transmission electron microscope.

[0036] 1.7. Detection of Reactive Oxygen Species (ROS)

[0037] Cerebral cortical sections were cut using a cryostat, rinsed with distilled water, and gently dried. Tissue areas were delineated using a tissue marking pen to prevent antibody diffusion. ROS detection dye was then applied within the marked circle, and the samples were incubated in a dark room at 37°C for 30 minutes. The slides were then immersed in PBS and rinsed three times on a staining rack. After the sections were gently dried, DAPI staining solution was applied within the circle and incubated in the dark at room temperature for 10 minutes. After this, the slides were rinsed with PBS on a rocking platform for three cycles, each lasting 5 minutes. After gently drying again, they were covered with anti-fading mounting medium. Finally, the slides were examined under a fluorescence microscope and images were captured. (DAPI has a UV excitation wavelength of 330-380 nm and an emission wavelength of 420 nm, emitting blue light; CY3 is excited at a wavelength of 510-560 and emits at 590 nm, emitting red light.).

[0038] 1.8. Glutathione peroxidase (GSH-Px), Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase detection

[0039] Frozen rat hippocampal tissue was collected, weighed, mixed with an appropriate amount of PBS, and then thoroughly homogenized on ice. After centrifugation, the supernatant was extracted. The activity of glutathione peroxidase (GSH-Px), sodium potassium ATPase (Na-ATPase) and glutathione peroxidase (GSH-Px) in hippocampal tissue was measured according to the provided protocol. + -K + -ATPase) and calcium-magnesium ATPase (Ca 2+ -Mg 2+ -ATPase) enzyme activity. The absorbance of these samples was measured at a wavelength of 450 nm using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader. Subsequently, a standard curve was constructed using the ELISA method to quantify GSH-Px, Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase levels.

[0040] Detection of autophagy proteins

[0041] A certain amount of hippocampal tissue was weighed, lysis buffer was added, and the cells were thoroughly homogenized. After centrifugation, the supernatant was collected, and protein concentration was determined using bicinchoninic acid (BCA). PBS was added to the sample buffer, mixed, and denatured in boiling water for 10 minutes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then immersed in 5% nonfat dry milk blocking solution for 2 hours at ambient temperature and rinsed with TBST after transfer. The membrane was incubated overnight at 4°C (with a dilution ratio of 1:1000 for PINK1, Parkin, COX IV, and P62; and a dilution ratio of 1:200 for LC3B). The membrane was then rinsed with TBST three times for 10 minutes each. Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were then added (dilution ratio of 1:2000, respectively). After film development, ECL staining was performed and the membrane was exposed in the dark for visualization. The relative expression level of the target protein was expressed as the ratio of its gray value to that of the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and statistical analysis was performed using Gelpro32.

[0042] 1.10. Detection of autophagy protein-related genes mPINK1, mParkin, mLC3B, mCOXⅣ, mP62, mBcl-2, mBax, mCyt-c, and mCaspase

[0043] Total RNA was extracted from the hippocampus using 1 mL of Trizol, and the concentration and purity of the RNA were measured. The forward and reverse sequences are as follows:

[0044] mPINK1 (forward) 5′-gaggagcagcaggcggagag-3′;

[0045] mPINK1 (reverse) 5′-tcgtgtgtctagtgggtcag-3′;

[0046] mParkin (forward) 5′-ctcagaccaggacacagtagc-3′;

[0047] mParkin (reverse) 5′-ggcggtggttacataggaagac-3′;

[0048] mLC3B (forward) 5′-gaggagcagcaggcggagag-3′;

[0049] mLC3B (reverse) 5′-tcgtgtgtctagtgggtcag-3′;

[0050] mCOXⅣ (forward) 5′-gccagcagtggcagaatgttg-3′;

[0051] mCOXⅣ(reverse)5′-cgagcgcacaccgaagtagaaatg-3′;

[0052] mP62 (forward) 5′-gagcagaggaagatagccttgg-3′;

[0053] mP62 (reverse) 5′-acttggagtcacctgtggatgg-3′;

[0054] mBcl-2 (forward) 5′-gagacgcgcgtgccacgac-3′;

[0055] mBcl-2 (reverse) 5′-gccagcgtcgcgaccagag-3′;

[0056] mBax (forward) 5′-gacgcaacctgagctgaccttg-3′;

[0057] mBax (reverse) 5′-catcggcatatcgcctgagac-3′;

[0058] mCyt-c (forward) 5′-agcagccaagcataagactggac-3′;

[0059] mCyt-c (reverse) 5′-tggtgtcatctgtgtaagagaatcc-3′;

[0060] mCaspase3 (forward) 5′-tggtgtcatctgtgtaagagaatcc-3′;

[0061] mCaspase3 (reverse) 5'-gccgtagactaagcatacaggaag-3'.

[0062] Amplification conditions: pre-denaturation at 95°C for 15 min; 40 cycles, each cycle including denaturation at 95°C for 34 seconds and annealing at 60°C for 34 seconds. GAPDH was used as the reference gene, and the expression level of the target gene was calculated by the 2-ΔΔCt method.

[0063] Statistical analysis

[0064] Data were statistically analyzed using SPSS 22. Data were normally distributed and presented as mean ± standard deviation. One-way analysis of variance was used to assess intergroup differences, with the LSD method used to test for homogeneity of variance and the Dunnett T3 test used to test for heterogeneity of variance. A P value < 0.05 was considered statistically significant.

[0065] 2. Results

[0066] 2.1. Basic information results

[0067] During the experiment, the rats' food intake, water intake, independent activity and mental state were observed every day. During the exposure period, the rats in the control and low-dose groups developed well, ate normally, moved freely, had smooth fur, and no abnormalities were found. As the exposure time increased, the rats in the medium and high-dose groups showed varying degrees of decreased activity, worsening mental state, worsening reaction ability, and the fur color of the high-dose group became dull. The data on body weight showed that there were substantial differences among the four groups (F = 18.78, P < 0.01), and the weight of the rats changed significantly over time (F = 305.07, P < 0.01). The interaction effect between treatment and exposure time was also shown to be statistically significant (F = 10.79, P < 0.01). The results of the interaction effect analysis are shown in Tables 1 and Figure 1 shown.

[0068] Table 1 Body weight of rats injected intraperitoneally with aluminum salicylate (x±S)

[0069]

[0070] Note: Control, low-dose, medium-dose, and high-dose groups. Mean ± SD. n = 8. *P < 0.05, **P < 0.01 compared with concurrent CG.

[0071] 2.2. Morris water maze test results

[0072] On days four and five of the study, rats in the high-dose group exhibited significantly longer escape latencies compared with the control and low-dose groups (P < 0.01). Within-group comparisons revealed that the escape latencies of rats in all groups were significantly shorter compared with the previous day (P < 0.05). In the spatial exploration test on day six, there were no differences in the total swimming distance within 120 seconds between the treatment groups (P > 0.05), but there were differences in the swimming distance within the target quadrant. Compared with the low-dose and control groups, the high-dose and medium-dose groups swam significantly shorter distances within the target quadrant (P < 0.05, P < 0.01, respectively). The number of rats crossing the platform and its respective quadrants was significantly reduced in the high-dose group compared with the control and low-dose groups (P < 0.01). Water maze trajectory diagrams showed that the swimming path of the control group became increasingly distinct over time. The path to the platform gradually became clear, with a largely linear trajectory. Although rats in the aluminum-exposed group eventually reached the platform, they exhibited disorganized and haphazard swimming trajectories. As the number of experimental days increased, the time required for rats to locate the platform gradually decreased, while as the amount of aluminum exposure increased, the time required for rats to locate the platform significantly increased. When the platform was removed, the frequency with which rats crossed the platform and its corresponding quadrant decreased as the exposure dose increased. The above behavioral test results show that maltol aluminum exposure reduces the ability of rats to explore new objects and their learning and memory functions, showing cognitive impairment in rats, such as Figure 2 、 Figure 3 shown.

[0073] 2.3. New Object Experiment Results

[0074] Results of the discrimination index indicated that the exposed group was less able to distinguish between novel and familiar objects than the control group. Furthermore, the high-dose group showed a significant decrease in novel object recognition compared to the control group, as well as the low- and medium-dose groups (P<0.05, P<0.05, and P<0.01). Preference index data indicated that memory abilities declined in all three treatment groups compared to the control group, with the high-dose group experiencing a significantly greater decrease (P<0.05, P<0.01).

[0075] Results of ROS and GSH-Px

[0076] The ROS detection results showed that with the increase of dose, the red fluorescence intensity in the rat hippocampus tissue increased significantly, indicating that the ROS level gradually increased. It is worth noting that the ROS level in the hippocampus tissue of rats in the high and medium dose groups increased significantly. At the same time, with the increase of dose, the GSH-Px content in the rat hippocampus gradually decreased, and the high dose group was significantly reduced. (P<0.01), such as Figure 4 shown.

[0077] 2.5. Mitochondrial electron microscopy results

[0078] The mitochondrial morphology of the control group was linear, and the cristae structure was intact and dense. The mitochondria in the low-dose group were slightly deformed and swollen, the cristae structure was slightly damaged, and the arrangement was loose. The mitochondria in the medium-dose group were deformed and swollen, and even vacuolated, with sparse cristae structure. Some damaged mitochondria were wrapped by double membranes to form mitochondrial autophagosomes. Most mitochondria in the high-dose group were microspherical, severely damaged, and the number of mitochondrial autophagosomes increased. Figure 5 shown.

[0079] 2.6.Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase results

[0080] The results showed that with the increase of maltodextrin aluminum exposure, rat hippocampal Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase activity gradually decreased. In addition, compared with the control group and the low-dose group, the ATPase activity of the medium- and high-dose aluminum exposure groups was significantly reduced (P < 0.01), such as Figure 6 shown.

[0081] 2.7. Results of proteins PINK1, Parkin, LC3B, COXⅣ, and P62 showed

[0082] As the exposure dose of maltol aluminum increased, the relative expression levels of PINK1 and Parkin also increased. The relative expression levels of PINK1 in the medium-dose and high-dose groups were significantly higher than those in the control and low-dose groups, and the differences were statistically significant (P<0.01, P<0.01); the relative expression level of Parkin in the high-dose group was significantly higher than that in the control and low-dose groups, and the differences were statistically significant (P<0.01, P<0.01). LC3B protein results showed that with the increase in aluminum exposure, the LC3B-II / LC3B-Ⅰ ratio increased significantly. The relative expression ratio of the high-dose group was significantly higher than that of the control group, and the difference was statistically significant (P<0.01). The high-dose group was significantly higher than the low-dose and medium-dose groups, and the differences were statistically significant (P<0.05, P<0.05). The relative expression levels of proteins P62 and COXⅣ gradually decreased with the increase in aluminum exposure. The relative expression levels of the medium-dose and high-dose groups were significantly lower than those of the control group, and the differences were statistically significant (P<0.05, P<0.05). Figure 7 shown.

[0083] 2.8. Results of autophagy-related proteins

[0084] As the exposure dose of maltol aluminum increased, the relative expression levels of mPINK1 and mParkin also increased. The relative expression levels of mPINK1 in the medium-dose and high-dose groups were significantly higher than those in the control group (P<0.01, P<0.01); the relative expression levels of mParkin in the medium-dose and high-dose groups were significantly higher than those in the control group and low-dose group, and the differences were statistically significant (P<0.05, P<0.01). The results of mLC3B showed that with the increase of aluminum exposure dose, its relative expression levels increased significantly, and the relative expression level in the high-dose group was significantly higher than that in the control group, low-dose group and medium-dose group, and the differences were statistically significant (P<0.01). The relative expression levels of mP62 and mCOXⅣ gradually decreased with the increase of aluminum exposure dose, and the relative expression levels in the medium-dose and high-dose groups were significantly lower than those in the control group, and the differences were statistically significant (P<0.01, P<0.01). Figure 8 shown.

[0085] 2.9. Apoptosis-related gene results

[0086] The data showed that the relative expression of Bax, cytochrome c, and Caspase-3 in the hippocampus of rats gradually increased, which was related to the increase in the exposure dose of maltol aluminum in different groups. The relative expression level of Bcl-2 gradually decreased with the increase in the exposure dose of maltol aluminum. Compared with the control group, the comparative levels of Bax, cytochrome c, and Caspase-3 in the group exposed to the highest dose of maltol aluminum were significantly increased, with statistical significance (P<0.05). In contrast, Figure 9 As shown, when juxtaposed with the control group, the expression of Bcl-2 in the high-dose group was significantly reduced, also reaching statistical significance (P<0.05).

[0087] 3. Discussion

[0088] Aluminum, a lightweight but toxic metal, is ubiquitous on Earth and has attracted considerable attention for its harmful effects. When ingested in high amounts, aluminum not only increases plasma aluminum levels but also distributes to various tissues, particularly the brain. Aluminum accumulation in the brain may cause neurotoxicity, leading to significant impairments in learning and memory. The novel object recognition test is a commonly used technique for assessing short- and long-term spatial learning and memory in rodents. Results from the novel object test in this study indicate that aluminum exposure impairs the cognitive abilities of rats in terms of learning and memory. With increasing aluminum exposure, the discrimination index results showed a downward trend, indicating a decrease in the ability of rats to learn novel objects and impaired learning and memory. Therefore, the preference index and discrimination index results suggest that aluminum exposure to maltodextrin impairs spatial learning and memory in rodents. Results from the Morris water maze test showed that on the fourth and fifth days of the location navigation test, the high- and medium-dose groups took significantly longer to locate the platform in the water than the control and low-dose groups. This study demonstrates that subchronic exposure to aluminum maltodextrin impairs spatial learning in rats. In the spatial exploration test on the sixth day, there was no difference in the total swimming distance in 120 seconds and no loss of motor ability, but the swimming distance in the platform quadrant decreased with increasing aluminum exposure concentration. Compared with the control group, the high-dose and medium-dose groups significantly reduced the number of swimming times across the platform position and the number of swimming times across the platform quadrant, indicating that subchronic aluminum exposure also impairs the rats' memory ability.

[0089] Oxidative stress refers to a situation in which the production of reactive oxygen species (ROS) in the body escalates in response to internal and external triggers, which exceeds the body's own clearance capacity. The disruption of the balance between pro-oxidant and antioxidant mechanisms leads to oxidative damage in the body. ROS is the main oxidative index.

[0090] Various studies have shown that aluminum exposure is associated with the generation of reactive oxygen species (ROS) and the induction of oxidative stress. Therefore, oxidative stress caused by ROS generation is considered a contributing factor to aluminum-related disease processes. Following aluminum exposure, ROS levels in the body increase, leading to oxidative damage. In this study, we examined the increase in ROS expression in the rat hippocampus following subchronic aluminum exposure. The results showed that with increasing dose, red fluorescence intensity in the rat hippocampus significantly increased, and ROS expression in the brain increased. Increased ROS may bind to fatty acids in the brain, leading to oxidative stress, protein, lipid, and nucleic acid oxidation, and enhanced oxidative damage. With increasing exposure dose, glutathione peroxidase (GSH-Px) levels in the rat hippocampus gradually decreased. GSH-Px is a widely present antioxidant kinase that protects against oxidative damage and is an indicator of antioxidant capacity. Decreased GSH-Px expression suggests that depletion of GSH-Px leads to an imbalance between redox homeostasis and oxidative stress. Some believe that long-term, low-level exposure to maltoaluminum can lead to an increase in ROS in the nervous system, stimulate the occurrence of oxidative stress, reduce the content of GSH-Px in the rat brain, unbalance the pro-oxidant and antioxidant capacity of brain tissue, and ameliorate the aggressive effects of ROS on tissues. Therefore, it is speculated that maltoaluminum-induced neuronal oxidative stress and antioxidant enzyme system disorders may be the mechanism of maltoaluminum neurotoxicity.

[0091] Mitochondria are the "powerhouses" of cells, generating the majority of cellular ATP through oxidative phosphorylation (OXPHOS), providing energy for cellular metabolism and material transformation, thereby maintaining cellular and bodily physiological activities. Mitochondrial damage is intricately linked to the excessive production of reactive oxygen species (ROS). Mitochondria produce ROS but are also susceptible to their effects. The results of this study showed that the expression of reactive oxygen species (ROS) in rat brain tissue increased with increasing doses of maltoaluminum. Electron microscopy revealed that in the control group, most mitochondria were elongated in shape, with intact and dense cristae. However, in the maltoaluminum-treated group, most mitochondria appeared swollen, with sparsely arranged cristae and perinuclear vacuoles. In the high-dose group, most mitochondria were lobulated, and the number of severely damaged mitophagosomes increased. This suggests that exposure to maltoaluminum leads to elevated ROS levels in rat brain tissue. Excessive ROS attack mitochondria, leading to mitochondrial structural damage and dysfunction. Damaged mitochondria may release more reactive oxygen species (ROS), exacerbating oxidative damage and further inhibiting mitochondrial function, creating a vicious cycle that leads to mitochondrial dysfunction and compromises mitochondrial structural integrity. The structural integrity of mitochondria is damaged, with swelling, vacuolization and cristae rupture. ATPase is a lipid enzyme that maintains the normal operation of the mitochondrial electrochemical gradient to maintain the mitochondrial membrane potential. + -K +-ATPase acts as a sodium-potassium pump, not only providing energy for sodium and potassium ions to cross the membrane potential, but also controlling the concentration of calcium ions. + -K + Inactivation of the ATPase leads to mitochondrial swelling and dissolution, resulting in an uncontrolled influx of sodium ions and efflux of potassium ions. 2+ -Mg 2+ -ATPase is responsible for supervising the transport of calcium and magnesium ions to ensure intracellular calcium homeostasis. When mitochondrial function and structure are damaged, Na + -K + -ATPase and Ka 2+ -Mg 2+ -ATPase is the most sensitive indicator. This study showed that Na + -K + -ATPase and Ka 2+ -Mg 2+ The activity of β-ATPase decreased with increasing doses, indicating that subchronic aluminum exposure can lead to a decrease in ATPase activity, which indirectly indicates a decrease in mitochondrial energy supply capacity. These results suggest that aluminum exposure to maltol may cause mitochondrial damage.

[0092] Excessive ROS can cause mitochondrial damage. When mitochondria are damaged, they are cleared through autophagy, a process known as mitophagy. The mechanisms by which autophagosomes degrade mitochondria are diverse, but the most well-known pathway is PINK1 / Parkin-induced mitophagy. When mitochondria are damaged, PINK1 rapidly accumulates in the outer mitochondrial membrane and activates mitophagy by recruiting Parkin from the cytoplasmic environment to the outer mitochondrial membrane. Parkin induces the aggregation of the junctional protein p62, which recruits the microtubule-associated protein LC3 and promotes its localization to depolarized mitochondria, leading to fusion of mitochondria with lysosomes to form mitophagosomes, which are then degraded. LC3 protein associated with microtubules is a biomarker of mitophagy. When autophagy is inactive, LC3B exists as the cytoplasmic soluble LC3B-I. When autophagy is activated, LC3B becomes activated. LC3B-I undergoes ubiquitin-like processing and binds to phosphatidylethanolamine on the autophagosome membrane to form LC3B-II. LC3B-II is located on the autophagosome membrane as a cluster and participates in autophagosome formation. Therefore, LC3B-II exhibits the highest accuracy in reflecting autophagic activity, with an increase in the LC3B-II / LC3B-I ratio indicating autophagic activation. P62 is a well-known and recognized selective autophagy receptor located on autophagosomes, and its content is regulated by the extent of mitophagy. P62 interacts with the core autophagy protein LC3 through its domains, leading to its clearance and degradation. As mitophagic activity increases, p62 is degraded. Therefore, when autophagy stabilizes, p62 protein expression gradually decreases. Therefore, the negative correlation between LC3B-II / LC3B-I and p62 levels is used to determine the intensity of autophagy. COXIV, located within the inner mitochondrial membrane, is a hallmark enzyme of mitochondrial oxidative phosphorylation. Results showed that with increasing doses of maltoaluminum exposure, the relative expression levels of PINK1 and Parkin and the ratio of LC3B-II / LC3B-I increased. However, the relative expression of P62 gradually decreased with increasing doses of maltoaluminum exposure, indicating that maltoaluminum exposure stimulates the PINK1 / Parkin autophagy pathway in rat hippocampal mitochondria. Simultaneously, LC3B-I protein is converted to LC3B-II, and P62 is reduced, which may promote the development of autophagy. While mild autophagy is essential for maintaining cellular life, overexpression of autophagy is abnormal and excessive, and overactivated autophagy severely damages the proper environment for cellular survival. In this experiment, COXIV protein also gradually decreased with increasing doses of maltoaluminum exposure. As a mitochondrial marker, COXIV can be used to quantify mitochondrial number. Therefore, maltoaluminum exposure damages mitochondria, inducing activation of the PINK1 / Parkin autophagy pathway, resulting in excessive mitophagy and a reduction in mitochondrial number.

[0093] Programmed cell death, or apoptosis, is a genetically regulated process of cellular self-destruction. Caspase-3, a member of the caspase family, is a key enzyme in apoptosis. Caspase-3 is associated with B-lymphocytic neoplasia 2 (Bcl-2) family cells. Bcl-2 is an anti-apoptotic protein, and Bcl-2-associated X protein (Bax) is a pro-apoptotic protein. Mitochondria can regulate apoptosis. Inactivation of Bcl-2 or stimulation of Bax exacerbates mitochondrial damage and cytochrome c (Cyt-c) translocation. Cyt-c is the primary pro-apoptotic protein stored in mitochondria. The results of this study showed that aluminum exposure increased the expression levels of Bax mRNA, Cyt-c mRNA, and Caspase-3 mRNA in the rat cerebral cortex, while decreasing the expression of Bcl-2 mRNA. When Bax is overexpressed, it antagonizes Bcl-2 and disrupts the mitochondrial membrane. This promotes the release of Cyt-c from the mitochondrial interior into the cytoplasm, ultimately activating Caspase-3 through various signaling pathways. Upon activation, Caspase-3 leads to chromatin condensation and nuclease activation, further promoting cell apoptosis. Decreased Bcl-2 gene expression indicates accelerated cell apoptosis and aggravated neuronal impairment. In summary, exposure to aluminum maltol induces neuronal apoptosis.

[0094] In summary, exposure to aluminum maltol can increase the production of ROS in the rat nervous system. Excessive ROS will attack mitochondria, stimulate oxidative stress, lead to mitochondrial structural damage and dysfunction, and reduce the energy supply capacity of mitochondria. At the same time, damaged mitochondria will trigger the PINK1 / Parkin-mediated autophagy mechanism, resulting in excessive mitochondrial autophagy, further reducing the number of mitochondria. Mitochondria play a core regulatory role in apoptosis, so aluminum maltol exposure can affect the apoptosis of rat nerve cells by destroying and changing mitochondrial structure and function and reducing the number of mitochondria in rat brain and nervous tissue. The main mechanisms are as follows. Figure 9 shown.

[0095] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. PINK1 / Parkin regulates mitochondria in cognitive dysfunction, characterized by: The PINK1 / Parkin can be used to prepare drugs for treating cognitive dysfunction caused by excessive intake of aluminum.

2. The use of PINK1 / Parkin in treating cognitive dysfunction by regulating mitochondria according to claim 1, wherein: The PINK1 / Parkin can regulate the mitochondrial autophagy mechanism.

3. The use of PINK1 / Parkin in treating cognitive dysfunction by regulating mitochondria according to claim 2, wherein: The PINK1 / Parkin can inhibit excessive mitochondrial autophagy caused by cognitive dysfunction due to aluminum exposure, maintain mitochondrial homeostasis in patients, inhibit excessive formation of ROS, and alleviate patients' cognitive dysfunction.

4. The use of PINK1 / Parkin in treating cognitive dysfunction by regulating mitochondria according to claim 1, wherein: The aluminum exposure increases the production of ROS in the nervous system. Excessive ROS attacks mitochondria, stimulates oxidative stress, causes mitochondrial structural damage and dysfunction, and reduces the energy supply capacity of mitochondria. Damaged mitochondria trigger the PINK1 / Parkin-mediated autophagy mechanism, resulting in excessive mitochondrial autophagy, further reducing the number of mitochondria and causing normal apoptosis of the patient's nerve cells, thereby alleviating the patient's cognitive dysfunction.

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